Methods of identifying synthetic transcriptional and translational regulatory elements, and compositions relating to same

Inactive Publication Date: 2007-02-27
THE SCRIPPS RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0031]Optionally, a reporter cassette useful for identifying a transcriptional regulatory element according to a method of the invention is a dicistronic construct that includes the nucleotide sequence encoding the first reporter molecule, and also includes a second nucleotide sequence enc

Problems solved by technology

However, it alone usually does not contain all the information necessary for the modulated expression of a gene in different contexts in the developing or behaving organism.
However, the rules by which regulatory elements function either by themselves or in combination with other elements in the many genes in which these elements are found are not well understood.
Currently,

Method used

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  • Methods of identifying synthetic transcriptional and translational regulatory elements, and compositions relating to same
  • Methods of identifying synthetic transcriptional and translational regulatory elements, and compositions relating to same
  • Methods of identifying synthetic transcriptional and translational regulatory elements, and compositions relating to same

Examples

Experimental program
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Effect test

Example

EXAMPLE 1

Selection of Synthetic Transcriptional Regulatory Elements

[0118]This example describes the preparation of a vector useful for selecting transcriptional regulatory elements and the identification and characterization of synthetic transcriptional regulatory elements.

[0119]A promoter element proviral vector library was constructed using the retroviral-mediated EGFP / FACS selection strategy for synthetic promoter elements according to the disclosed methods. A library of promoter elements (random 18 mers; Ran18) was constructed in the proviral selection vector, which was packaged into retroviral particles in COS1 cells. The retroviral particles were harvested and used to infect target cells, which were then treated for 3 days with puromycin to kill uninfected or poorly expressing cells. The surviving cells were subjected to FACS analysis and the most highly fluorescent cells collected. Genomic DNA was prepared from these cells and the regulatory oligonucleotides were recovered by...

Example

EXAMPLE 2

Validation of Synthetic Regulatory Element Selection Method

[0164]This example demonstrates the disclosed synthetic regulatory element selection method can be used routinely to screen libraries of oligonucleotides and can consistently identify synthetic transcriptional regulatory elements.

Example

[0165]The retrovirus vector MESVR / EGFP* / IRES / pacPro(ori) (SEQ ID NO: 1; see Example 1B) was used to screen a second library of Ran 18 sequences using the synthetic promoter construction method (SPCM). More than 100 DNA sequences that showed increased promoter activity (4 to 50-fold) in the neuroblastoma cell line Neuro2A were identified. The DNA sequences of selected synthetic promoters were determined and database search using the RIGHT software package, which allowed simultaneous comparison of a database of active Ran 18 elements to existing databases such as TransFac. The search revealed a predominance of eight motifs—AP2, CEBP, GRE, Ebox, ETS, CREB, AP1, and SP1 / MAZ; about 5 to 10% of the active DNA sequences were not represented in known transcription factor databases and appeared to be novel. The most active of the selected synthetic promoters contained composites of pairs, triples, or quadruples of these motifs. Assays of DNA binding and promoter activity of three exemplary m...

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Abstract

Provided are methods of identifying oligonucleotides having transcriptional or translational activity by integrating the oligonucleotide into a eukaryotic cell genome such that the oligonucleotide is operatively linked to an expressible polynucleotide, and detecting a change in expression of the expressible polynucleotide due to the operatively linked oligonucleotide. Also provided are vectors useful for identifying an oligonucleotide having transcriptional or translational regulatory activity according to a method of the invention. In addition, isolated synthetic transcriptional or translational regulatory elements identified according to a method of the invention are provided, as are kits, which contain a vector useful for identifying a transcriptional or translational regulatory element, or an isolated synthetic transcriptional or translational regulatory element or plurality of such elements. Also provided are isolated transcriptional regulatory elements.

Description

[0001]This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Ser. No. 60 / 230,956, filed Sep. 7, 2000; U.S. Ser. No. 60 / 230,852, filed Sep. 7, 2000; U.S. Ser. No. 60 / 207,804, filed May 30, 2000; U.S. Ser. No. 60 / 186,496, filed Mar. 2, 2000; U.S. Ser. No. 60 / 178,816, filed Jan. 28, 2000; and U.S. Ser. No. 60 / 261,312, filed Jan. 12, 2001, each of which is incorporated herein by reference.[0002]This invention was made in part with government support under Grant No. MCB9982574 awarded by the National Science Foundation. The government has certain rights in this invention.BACKGROUND OF THE INVENTION[0003]1. Field of the Invention[0004]This invention relates to methods for producing nucleotide sequences having regulatory functions using cellular selection of random nucleotide sequences, and to the sequences so produced.[0005]2. Background Information[0006]Every eukaryotic gene has a core promoter that resides at the extreme 5′ end of its transcription unit. Most c...

Claims

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Application Information

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IPC IPC(8): C07H21/02C07H21/04C12N15/00C12N15/09C12N15/63C12N15/70C12N15/74C12N15/10C12N15/79C12N15/867C12N15/90C12Q1/68
CPCC12N15/1048C12N15/1051C12N15/67C12N15/86C12N15/90C12N2840/203C12N2740/13043C12N2800/108C12N2830/002C12N2830/15
Inventor MAURO, VINCENT P.EDELMAN, GERALD M.CHAPPELL, STEPHEN A.JONES, FREDERICK S.OWENS, GEOFFREYMEECH, ROBYN
Owner THE SCRIPPS RES INST
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